JP2002503642A - Pharmaceutical composition comprising long pentraxin PTX3 - Google Patents

Abstract

(57) Abstract: A pharmaceutical composition for the treatment of infectious and inflammatory or neoplastic diseases, comprising long pentraxin PTX3, especially human PTX3; an expression vector comprising a cDNA encoding PTX3; Disclosed are recombinant host cells transfected with; methods for producing substantial quantities of PTX3, including cultures of such cells, and the use of the vectors in gene therapy of tumors.

Description

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a pharmaceutical composition comprising long pentraxin PTX3 (PTX3), or one of its functional derivatives. In particular, the present invention relates to such a composition for the treatment of infectious and inflammatory diseases or tumors.

[0002] The present invention also relates to an expression vector comprising a complete cDNA encoding PTX3 or one of its functional derivatives, a recombinant host cell transfected with such an expression vector, and PTX3 or The present invention relates to a method for producing one of the functional derivatives. Furthermore, the invention relates to a method of gene therapy based on the use of said expression vector for the treatment of tumors.

After exposure to the inflammatory cytokines interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha), various types of cells, most notably mononuclear, To date, the biological function of PTX3, a protein expressed in phagocytes and endothelial cells, has not been fully understood to date.

[0004] To date, no therapeutic use of PTX3 or its functional derivatives has been reported.

[0005] PTX3 consists of two structural regions, the N-terminus is not associated with any known molecule, and the C-terminus is similar to short pentraxins such as C-reactive protein (CRP). A considerable degree of similarity has been found between human PTX3 (hPTX3) and animal PTX3.

[0006] The PTX3 gene is present in a region similar to the human 3q region (q24-28) on mouse third chromosome, consistent with the position of hPTX3 reported to be in the 3q25 region. . Furthermore, mouse PTX3 (mPTX3) (Introna M., Vidal
Alles V., Castellano M., Picardi G., De Gioia L., Bottazzi B., Peri G.,
Breviario F., Salmona M., De Gregorio L., Dragani TA, Srinivasan N.,
Blundell TL, Hamilton TA and Mantovani A .: Cloning of mouse PTX3, a new member of the pentraxin gene family expressed in extrahepatic sites. Bl
ood 87 (1996) 1862-1872) is very similar to hPTX3 in composition, position and sequence (Breviario F., d'Aniello EM, Golay J., Peri G., Bottazzi
B., Bairoch A., Saccone S., Marzella R., Predazzi V., Rocchi M., Della
Valle G., Dejana E., Mantovani A., Introna M .: Interleukin-1-inducible gene in endothelial cells. Cloning of novel genes related to C-reactive protein and serum amyloid P component. J. Biol. Chem. 267: 22190, 1992).

In particular, the degree of identity between sequences is 82% between human and mouse genes and reaches 90% when conservative substitutions are considered.

[0008] The high degree of identity between the hPTX3 and mPTX3 sequences is a sign of a high degree of conservation of pentraxin during evolution (Pepys MB, Baltz ML: C-reactive protein and related proteins (pentraxin). Adv. Immunol. 34: 141, 1983) and acute phase response proteins specifically related to serum amyloid A protein.

[0009] CRP is a marker for immune-inflammatory and infectious diseases. Its purpose was to prevent the damage from spreading or being damaged by tissue damage or infection after trauma, destroy the infected organism, and activate repair mechanisms to restore normal functioning. A complex series of reactions is elicited in affected patients. This process constitutes the so-called acute phase response, but the main marker currently used for the acute phase response is CRP. In normal human serum, CRP is usually 10
It is present at concentrations less than μg / ml, but may increase by over 1,000-fold in response to trauma or inflammation (Koj A .: “Acute phase reactants” in “Structure a
nd Function of Plasma Proteins ". Allison A., ed. Plenum Press, New York,
1974, pp. 73-131).

Previous therapeutic uses of CRP are already known. For example, 1
US Patent 4,857,314, issued August 15, 989, discloses the use of CRP in combination with TNF for the treatment of tumors.

International Patent Application No. PCT / US94 / 02181, filed Feb. 24, 1994,
Disclosed are the preparation of diagnostic kits for the measurement of immune complexes in biological fluids and variants of CRP useful for the treatment of viral and microbial diseases, tumors and endotoxin shock.

International Patent Application No. PCT / US94 / 09729, filed August 26, 1994, also discloses variants of CRP useful for preparing diagnostic kits and treating viral and microbial diseases and tumors.

PTX3 recognizes the ligand recognized by short pentraxin,
And the ability to specifically bind has been evaluated in vitro using purified recombinant PTX3. Short pentraxins, such as CRP and SAP (serum amyloid P component), are characterized by their ability to recognize and bind in a calcium-dependent manner to a wide range of ligands, including phosphocholine, phosphoethanolamine, and many carbohydrates. The most characteristic of these are pyruvate-rich agarose derivatives, [methyl 4-6-O- (1-carboxyethylidene) -beta-D-galacto-pyranoside] or MOβDG, complement cleavage. Fragments and extracellular matrix proteins, especially fibronectin and type IV collagen. Unlike short pentraxin, PTX3 cannot bind to calcium or phosphocholine (as measured by inductively coupled plasma / atomic emission spectrometry), either phosphoethanolamine or MOβDG.
Furthermore, PTX3 cannot bind extracellular matrix proteins such as fibronectin or type IV collagen. On the other hand, PTX3 can bind to the C1q complement fragment, which is also recognized by short pentraxin (Table 1). However, it has been emphasized that CRP and SAP must be cross-linked in order to bind C1q, whereas PTX3 can recognize and bind this complement fragment in its naturally existing form. It should be.

Surprisingly, it has now been found that long pentraxin PTX3 or a functional derivative thereof is a useful therapeutic agent, especially for the treatment of infectious and inflammatory diseases or tumors.

What is meant by “long pentraxin PTX3” is long pentraxin PTX
It can be any of X3, i.e., whether of natural (human or animal) or synthetic origin. Human long pentraxin PTX3 (see sequence 1 and FIG. 5) is the preferred form.

[0016] A convenient method for producing full-length, long pentraxin PTX3 or one of its functional derivatives is to use an expression vector comprising the complete cDNA sequence encoding PTX3 or one of its functional derivatives (eg, Plasmids) and transfecting them into cultured eukaryotic cells (eg, Chinese hamster ovary cells, CHO). After cloning the transfected recombinant host cells, cell clones capable of producing the highest levels of PTX3 are selected.

According to the present invention, the above-mentioned expression vector comprising the cDNA sequence encoding the long pentraxin PTX3 is also used in gene therapy for the treatment of neoplastic diseases.

[0018] The first method of gene therapy is: a) taking a sample of cells from a patient affected by a tumor; b) a complete cDNA encoding long pentraxin PTX3 or one of its functional derivatives Transfecting these cells with an expression vector comprising the sequence,
And c) inoculating these transfected cells into a tumor patient.

A second method of gene therapy for the treatment of tumors comprises: a) containing a complete cDNA sequence encoding a long pentraxin PTX3 or one of its functional derivatives (such as an adenovirus or retrovirus). Na)
Preparing an expression vector of viral origin; and b) inoculating the expression vector thus obtained into a patient affected by a tumor.

Although the mechanism of action of PTX3 or a functional derivative thereof has not yet been elucidated, at least its anticancer effect is due to a direct cell lysis or cytostatic effect on tumor cells. Rather, it is due to a host-mediated mechanism and a mechanism related to the leukocytosis potential exerted by these compounds, as described below.

The following describes examples and reports results illustrating the unexpected effects of the compounds according to the invention described therein.

Production of recombinant PTX3: A fragment containing the complete cDNA sequence of human PTX3 (SEQ ID NO: 2 and FIG. 6) was subcloned into the BamH1 site of the expression vector pSG5 (FIG. 1) (Stratagene, La Jolla, CA, USA), transfected CHO cells using the precipitated calcium method. Proteins were purified by ion exchange and gel filtration chromatography from clones that were selected in G418 and capable of producing large amounts of PTX3 as a source.

[0023] Binding of PTX3 to C1q: Binding of PTX3 to C1q was measured by an ELISA system. The 96-well plate is covered with 250-500 ng C1q per well (overnight, 4 ° C.) and then Ca containing 0.05% Tween 20
⁺⁺ and Mg ⁺⁺ containing PBS (PBS). Well then P
Block with 5% milk in BS (2 hours room temperature), then add various concentrations of PT
Incubated with X3 (30 minutes at 37 ° C). After washing several more times, PTX
Plates were incubated with rat monoclonal antibody to 3 (1 hour at room temperature) and then with a second antibody, peroxidase-conjugated rat anti-IgG antibody (1 hour at room temperature). After washing, chromogen was added and the absorbance was read at 405 nm using an automatic plate reader. In some experiments, wells were covered with PTX3 and C1q binding was assessed using an anti-C1q antibody.

[0024] The biotinylated protein was used to determine the binding affinity of C1q. PTX3 was biotinylated (SPA-Societa Prodotti Antibiotici) according to a conventional method using activated biotin modified by adding a “spacer arm”.

FIGS. 2 (A) and 2 (B) show the results of C1q binding and binding affinity. These results indicate a very high C1q binding and binding affinity of PTX3.

Leukocyte increase: PTX3-induced leukocyte increase was investigated in vivo in a subcutaneous pocket system. A subcutaneous pocket was induced in the experimental animals by two subcutaneous injections of 5 mL of air into the back of the animal at treatment intervals of 3 days.
On day 6, 1 μg of PTX3 in 0.5% carboxymethylcellulol was administered to the pocket. Four hours later, the animals were anesthetized and the pockets were washed with 1 mL of saline solution. The wash was collected and the total number and fraction of cells present were counted.

The results obtained are reported in FIG. 3, which demonstrates the substantial leukocytosis potential of PTX3 in normal animals. On the other hand, FIG. 4 shows the results obtained in genetically engineered animals without C1q, but the leukocyte increase is clearly lower.

Anticancer activity: Mouse mast cell tumor P815 strain was co-transfected by electroporation with expression vector pSG5 containing cDNA of human PTX3 or its antisense and vector pSV2 which imparts neomycin resistance to transfected cells. . After selection with 0.5 mg / mL neomycin, cells were cloned by limiting dilution.

To measure PTX3 production, 2.5 × 10 ⁵ cells were cultured in 200 μl of RPMI + 3% FCS for 24 hours and the supernatant was tested by ELISA. The resulting clones produced levels of protein ranging from 1 to 35 ng / mL, whereas clones containing antisense did not produce measurable levels of PTX3. The clones studied showed the same growth rate in vivo.

8-10 week old male DBA / 2N CrlBR mice were inoculated subcutaneously with 1 × 10 ⁵ cells of a P815 PTX3-producing clone or a clone containing the antisense gene. Mice were monitored three times a day for tumor development and once a day for survival.

The results obtained are reported in Table 2, which shows that in this experimental model of gene therapy, PTX3 leads to healing of animals and complete tumor rejection after engraftment of inoculated tumor cells. It was to show.

These results are statistically significant at p <0.01 when compared to control and to the group treated with antisense (Fisher test).

In light of these results, it is clear that the previously reported anticancer effects are closely associated with the increase in leukocytes that occur in mice as a result of PTX3 recognizing C1q. In genetically engineered mice, no such leukocyte enrichment occurs. The leukocytosis potential is based on the anticancer activity of the compounds according to the invention, which suggests that these compounds may be effectively applied in the treatment of diseases caused by pathogens such as bacteria, fungi, protozoa or viruses. Suggests.

[0034]

Table 1 Table 1 Binding ability of pentraxin to various ligands ND: Not tested

[0035]

[Table 2] Table 2 PTX3 in vivo anti-cancer effect The indicated clones of 1: 1 × 10 ⁵ cells were injected subcutaneously. 2: Number of animals that completely rejected the tumor, relative to the total number of animals that survived the tumor. ^* : Compared to both mice treated with parental cells and mice treated with cells of the antisense clone. P <0.01 (Fisher test).

[0036]

[Sequence list]

[Brief description of the drawings]

FIG. 2. Binding of PTX3 to C1q. Panel A shows binding of culture supernatant and purified protein containing PTX3 (sense) to C1q and C1s immobilized on the plate. Binding was evaluated as the optical density (OD) at 405 nm. Panel B shows the saturation curve obtained with the biotinylated protein.
Kinetic parameters were calculated using non-linear fit statistics.

FIG. 3: PTX3-induced increase in leukocytes: CD1 mice are injected with 1 μg of PTX3 into the subcutaneous pocket induced on the back by inoculating 5 ml of air.

FIG. 4. PTX3-induced increase in leukocytes in normal and genetically engineered animals without C1q. PTX3 is injected subcutaneously induced on the back of the animal.

FIG. 5: Sequence 1 : amino acid sequence of human PTX3. Underlined amino acids constitute the peptide signal. Mature hPTX3 consists of 364 amino acids.

[6] Sequence 2: Nucleotide sequence of the cDNA fragment of human PTX3.
Uppercase letters indicate nucleotides encoding the protein, while lowercase letters indicate the 3 'and 5' regions that are not translated but are present in the construct.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) A61P 31/04 A61P 31/12 4H045 31/12 35/00 35/00 C12P 21/02 C C12N 5/10 (C07K 14/47 15/09 ZNA C12R 1:91) C12P 21/02 A61K 37/02 // (C07K 14/47 C12N 5/00 B C12R 1:91) 15/00 ZNAA (81) Designated country EP ( AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), EA (AM, AZ, B) , KG, KZ, MD, RU, TJ, TM), AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE , ES, FI, GB, GE, GH, GM, HR, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US , UZ, VN, YU, ZW (72) Inventor Martino Introna, Italy-20017 Rho (Milan), Via Barzalotti 12 / E number (72) Inventor Alberto Mantobani Italy, I-20100 Milan, Largo Brasilia No. 4 (72) Inventor Anne Ciata Becchi Italy, A-20131 Milan, Via Spontini 11th F-term (reference) 4B024 AA01 BA80 CA04 DA02 EA02 GA11 HA17 4B064 AG01 CA10 CA19 CC24 CE07 CE11 DA01 DA05 DA08 4B065 AA90X AA95X AA97X AB01 BA02 CA24 A44A08 BA01 BA22 MA52 MA55 MA63 NA14 ZB111 ZB261 ZB311 4C087 AA01 BC83 CA12 MA52 MA55 MA63 NA14 ZB11 ZB26 ZB31 4H045 AA20 AA30 BA10 CA40 EA22 EA28 EA29 FA74

Claims (18)

[Claims] 1. A pharmaceutical composition capable of oral, parenteral, transdermal or subcutaneous administration comprising an amino acid sequence of long pentraxin PTX3 as an active ingredient and a pharmacologically acceptable excipient. 2. The composition of claim 1, wherein the long pentraxin PTX3 sequence is a naturally occurring PTX3 sequence. 3. The composition of claim 2, wherein the long pentraxin PTX3 sequence is a human PTX3 sequence. 4. The composition according to claim 1, for treating infectious and inflammatory diseases or tumors. 5. The composition according to claim 4, for the treatment of a disease caused by bacteria, fungi, protozoa or virus. 6. An expression vector comprising the complete cDNA sequence encoding long pentraxin PTX3 or one of its functional derivatives. 7. The vector according to claim 6, which is a plasmid. 8. The vector according to claim 7, which is a plasmid pSG5. 9. A recombinant host cell transfected with the expression vector of claim 6-8. 10. The host cell according to claim 9, which is a CHO cell. 11. A method for producing long pentraxin PXT3 or one of its functional derivatives, comprising culturing the cell of claim 9-10. 12. A method comprising: (a) obtaining a sample of cells from a patient having cancer; (b) comprising the complete cDNA sequence encoding long pentraxin PXT3 or one of its functional derivatives. Transfecting said cells with an expression vector; and (c) inoculating said patient with said transfected cells: a method of gene therapy for the treatment of a neoplastic disease. 13. A preparation of an expression vector of viral origin comprising the complete cDNA sequence encoding the long pentraxin PXT3 or one of its functional derivatives; and (b) the expression vector thus obtained. Injection into patients with cancer: Gene therapy for the treatment of neoplastic diseases, including: 14. The method according to claim 13, wherein the expression vector derived from a virus is an adenovirus or a retrovirus. 15. Use of long pentraxin PTX3 for the preparation of a medicament for the treatment of infectious, inflammatory or neoplastic diseases. 16. Use according to claim 15, wherein the long pentraxin PTX3 is a human pentraxin having sequence 1. 17. Use according to claim 16, for the preparation of a medicament for the treatment of a disease caused by bacteria, fungi, protozoa or viruses. 18. A method for preparing an expression vector for use in a gene therapy method for treating a neoplastic disease, comprising an cDNA encoding a long pentraxin PTX3, or one of its functional derivatives. Use of cDNA.